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In-vivo imaging tracking system of mouse hepatitis coronavirus and application of tracing system
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A technology of coronavirus and vaccinia virus vectors, which is applied in the direction of viruses/bacteriophages, microorganism-based methods, and the use of vectors to introduce foreign genetic materials, etc., to achieve the effect of stable fluorescence quality
Active Publication Date: 2015-02-11
INST OF PLA FOR DISEASE CONTROL & PREVENTION
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[0005] There is no report on Gaussia luciferase tracing of murine hepatitis coronavirus
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Embodiment 1
[0068] The construction of the recombinant murine hepatitis coronavirus of embodiment 1, LUC mark
[0069] 1. Construction of Homologous Recombination Plasmids
[0070] 1. Construction and identification of recombinant plasmid pGPT-IN-ORF4
[0071]No. 27500-27966 (corresponding to No. 1-467 of Sequence 1) of the genomic cDNA sequence of wild-type murine hepatitis coronavirus A59 strain (GenBank number: NC_001846.1, Up date: 2012-8-23) was named upstream homology arm) and positions 28265-28700 (corresponding to positions 766-1201 of sequence 1, named downstream homology arms) were cloned into the upstream and downstream of the gpt gene of plasmid pSV2-gpt, respectively, to obtain the recombinant plasmid pGPT-IN- ORF4. The specific operation is as follows:
[0072] (1) Extraction of vaccinia virus inf-1 genomic DNA
[0073] Since the vaccinia virus vector vaccinia virus inf-1 contains the full-length cDNA of the genome of the wild-type mouse hepatitis virus (MHV) A59 strain,...
Embodiment 2
[0184] Embodiment 2, the biological activity detection of MHV-LUC recombinant virus
[0185] 1. Determination of one-step growth curve of MHV-LUC recombinant virus
[0186] The MHV-LUC recombinant virus prepared in Example 1 was used to infect (MOI=1) 17Cl-1 cells, and after adsorption at 37°C for 1 hour, the virus solution was removed, and the cells were washed with DMEM for 3 times to remove residual uninfected virus, and then in Cells were cultured at 33°C with DMEM containing 10% (volume percent) FBS. The virus was collected at different time points (0, 2, 4, 6, 8, 10, 12, 24 hours) within 0-24 hours after infection, and the collected virus was subjected to plaque analysis at 33°C to determine the virus titer (PFU / ml ), and then draw a one-step growth curve. At the same time, the wild-type MHV A59 strain was used as a control to detect the difference between the MHV-LUC recombinant virus and its one-step growth curve. Experiments were repeated three times.
[0187] The...
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Abstract
The invention discloses a LUC (Luciferase) tracing system of a mouse hepatitis coronavirus and application of the tracing system. The in-vivo imaging tracking system of mouse hepatitis coronavirus is used for recombining the mouse hepatitis coronavirus by replacing or inserting wild mouse hepatitis coronavirus genome RNA (Ribose Nucleic Acid); in the replacing process, any segment in a segment a in the wild mouse hepatitis coronavirus genome RNA is replaced with a segment b; in the inserting process, any locus in the segment a in the wild mouse hepatitis coronavirus genome RNA is inserted in the segment b; the segment a is RNA coded by a sequence l in a sequence table; and the segment b is RNA coded by a DNA (Deoxyribonucleic Acid) segment containing a caussia luciferase coding gene. According to the in-vivo imaging tracing system of the house hepatitis coronavirus and application of the tracing system disclosed by the invention, the MHV (Mouse Hepatitis Virus)-LUC and the wild virus can be used for research at the same time. Besides, the MHV-LUC can be used for greatly improving the virus monitoring flexibility due to an LUC signal amplifying effect, so that a new idea is provided for establishment of a low-dose virus subclinical infected animal model, and a new technical platform is provided for the application, research and the like of the coronavirus resistant drug screening.
Description
technical field [0001] The invention relates to a mouse hepatitis coronavirus live imaging tracing system and application thereof, in particular to a recombinant mouse hepatitis coronavirus obtained by inserting a Gaussia luciferase gene into the genome of the wild type mouse hepatitis coronavirus A59 strain. Background technique [0002] Coronavirus is the single-stranded positive-sense RNA virus with the largest genome known so far. It mainly causes diseases of the respiratory tract and digestive tract. It is the pathogen of various economic animal infectious diseases and often causes huge social and economic losses. Since a new SARS-CoV was identified as the causative agent of severe infectious disease SARS in 2003, coronavirus (Coronavirus) has become one of the hotspots of global life science research. [0003] Bioluminescent tracer technology is a new molecular and gene expression analysis and detection technology developed in recent years. In the field of molecular b...
Claims
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